Cross-linked bioprosthetic tissue using bio-orthogonal binding pairs

ABSTRACT

Methods for treating a bioprosthetic tissue and treated bioprosthetic tissue are described. The methods comprise contacting the biological tissue with an anchor compound, the anchor compound comprising first and second functional groups. The first functional group is reactive with and couples a tissue functional group associated with the biological tissue. The second functional group is one of a bio-orthogonal binding pair. The biological tissue coupled to the anchor compound is then exposed to a linking compound. The linking compound comprises at least two functional groups, each comprising the other one of the bio-orthogonal binding pair. In a preferred embodiment, the bio-orthogonal binding pair is an azide and an acetylene. The method can be performed in the presence of a catalyst, preferably a copper catalyst. Alternatively, the method can be performed in the absence of a catalyst, wherein the acetylene is incorporated in a ring-strained cyclic compound, such as cyclooctyne.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/936,097, filed Mar. 26, 2018, which is a continuation of U.S. patentapplication Ser. No. 14/078,435, filed Nov. 12, 2013, now U.S. Pat. No.9,925,303, which claims the benefit of U.S. Provisional PatentApplication No. 61/725,937, filed on Nov. 13, 2012, the contents all ofwhich are incorporated by reference in their entireties.

FIELD OF THE INVENTION

The present invention is directed to methods for treating bioprosthetictissue for implantation in a patient and, more particularly, to methodsfor cross-linking bioprosthetic tissue using bio-orthogonal bindingpairs.

BACKGROUND

Significant challenges are presented by the use of non-autologous tissuein bioprosthetic implants. Chief among the challenges are immunologicalrejection and/or calcification of the bioprosthetic implant which, inturn, results in the undesirable degradation and stiffening of thetissue. Immunological rejection and calcification are particularlyproblematic for bioprosthetic heart valves, as calcification of theseleaflets after implantation will adversely affect the leaflets' abilityto maintain the required one-way flow of blood, i.e., preventundesirable leaking or regurgitation of blood.

Glutaraldehyde has long been the reagent of choice for cross-linkingbiological tissues and, more particularly, for cross-linking pericardialtissue used for heart valves. Glutaraldehyde chemically modifies andcross-links collagen to render the biological tissue immunologicallyacceptable in the human host and stabilize the tissue. Whileglutaraldehyde remains the preferred cross-linking reagent, it is notwithout its disadvantages. Indeed, glutaraldehyde has been reported toaccelerate the calcification process, which is the main cause oflong-term failure in glutaraldehyde-fixed pericardial valves.Furthermore, as glutaraldehyde is cytotoxic and prevents host cellattachment, migration and proliferation, it hinders the ability oftreated tissue to regenerate in vivo. Glutaraldehyde also has a tendencyto polymerize and to produce undesired side reactions. The types ofreactions that are implicated by glutaraldehyde are often difficult tocontrol.

What is therefore needed are strategies that can be used in place of orin conjunction with glutaraldehyde fixation that mitigate some of thedisadvantages of glutaraldehyde-treated bioprosthetic tissue.

BRIEF SUMMARY

The preferred embodiments described herein are directed to methods fortreating biological tissue for use in connection with an implantablebioprosthesis.

In one preferred embodiment, a method for cross-linking biologicaltissue is described. The method comprises contacting the biologicaltissue with an anchor compound comprising first and second functionalgroups. The first functional group couples a tissue functional groupassociated with the biological tissue and the second functional group isone of a bio-orthogonal binding pair. The biological tissue is thenexposed to a linking compound comprising at least two functional groups.The two functional groups each comprise the other one of thebio-orthogonal binding pair.

In accordance with a first aspect, the bio-orthogonal binding paircomprises an azide and an acetylene.

In accordance with a second aspect, the exposing is performed in thepresence of a catalyst. The catalyst can be a copper, a ruthenium, asilver, salts of copper, ruthenium or silver, or derivatives of copper,ruthenium or silver. In a preferred embodiment, the catalyst is copper,a copper salt or derivatives of copper.

In accordance with a third aspect, the method further comprises rinsingthe biological tissue after exposing the biological tissue with thelinking compound. The rinsing can be performed using an aqueous,non-aqueous or anhydrous solution. Aqueous solutions include a salinesolution, preferably a buffered saline solution, such as aphosphate-buffered saline solution. Aqueous, non-aqueous or anhydroussolutions include glycerol solutions, polyethylene glycol (PEG)solutions, and ketone solutions, such as acetones.

The term “non-aqueous,” as it refers to a solution, is understood tomean a solution in which less than 50% by weight of the solution systemis water. Thus, a non-aqueous solution does not exclude the presence ofwater, either as an impurity or in amounts less than 50% by weight.

In accordance with a fourth aspect, the acetylene is incorporated in acyclic compound having a ring strain. In a preferred embodiment, thecyclic compound is a cyclooctyne. The cyclooctyne can comprise one ormore electron-withdrawing groups, preferably a halogen and mostpreferably a fluorine.

In accordance with a fifth aspect, the exposing is performed in theabsence of a catalyst.

In accordance with a sixth aspect, the tissue functional group is one ormore selected from the group consisting of an amine, a hydroxyl, asulfhydryl, a carbonyl, and a carboxylic acid. The tissue functionalgroup is preferably an amine and the first functional group of theanchor compound is an aldehyde.

In accordance with a seventh aspect, the first functional group of theanchor compound is selected from the group consisting of anisothiocyanate, an isocyanate, a sulfonyl chloride, an aldehyde, acarbodiimide, an acyl azide, an anhydride, a fluorobenzene, a carbonate,an N-Hydroxysuccinimides (NHS), an NHS ester, an imidoester, an epoxide,a fluorophenyl ester, an amine, a carboxylic acid, and an alcohol.

In accordance with an eighth aspect, the anchor compound is one or acombination of an imidazole-1-sulfonyl azide andtrifluoromethanesulfonyl azide.

In accordance with a ninth aspect, either one or both of the anchor andthe linking compounds comprises a spacer.

In accordance with a tenth aspect, the spacer does not comprisefunctional groups that are reactive with the biological tissue, with thetissue functional group or any one of the bio-orthogonal binding pair.

In accordance with an eleventh aspect, the linking compound comprisesthe spacer. The spacer can be one or a combination selected frombranched or straight-chain saturated or unsaturated hydrocarbons and apolymer. The spacer can also or additionally comprise one or acombination of a bioactive and a biodegradable group. The biodegradablegroup can be a disulfide.

In another embodiment, a cross-linked bioprosthetic tissue produced inaccordance with any one of the foregoing methods is provided.

In accordance with a first aspect, the cross-linked bioprosthetic tissueis not treated with glutaraldehyde, formaldehyde, or otheraldehyde-containing crosslinker.

In accordance with a second aspect, the cross-linked bioprosthetictissue is provided in a sealed package that does not contain a liquidpreservative solution in contact with the tissue.

Other objects, features and advantages of the described preferredembodiments will become apparent to those skilled in the art from thefollowing detailed description. It is to be understood, however, thatthe detailed description and specific examples, while indicatingpreferred embodiments of the present disclosure, are given by way ofillustration and not limitation. Many changes and modifications withinthe scope of the present disclosure can be made without departing fromthe spirit thereof, and the disclosure includes all such modifications.

BRIEF DESCRIPTION OF THE DRAWINGS

Illustrative embodiments of the present disclosure are described hereinwith reference to the accompanying drawings, in which:

FIG. 1 depicts the copper-catalyzed azide-alkyne cycloaddition reactionscheme.

FIG. 2 depicts the azide-cyclooctyne cycloaddition reaction scheme.

FIG. 3 depicts an embodiment of a ring-strained linking compound.

FIG. 4 depicts exemplary embodiments of functional groups which arereactive with, and thus couples with a tissue functional groupassociated with a biological tissue.

FIGS. 5A-5B depicts an exemplary method in which the anchor and linkingcompounds comprising the bio-orthogonal binding pair effectuatescross-links between tissue collagen fibers.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Specific, non-limiting embodiments of the methods for cross-linkingbioprosthetic tissue will now be described with reference to thedrawings. It should be understood that such embodiments are by way ofexample only and merely illustrative of but a small number ofembodiments within the scope of the present disclosure. Various changesand modifications obvious to one skilled in the art to which the presentdisclosure pertains are deemed to be within the spirit, scope andcontemplation of the present disclosure as further defined in theappended claims.

The preferred embodiments described herein are directed to methods fortreating biological tissue for use in connection with an implantablebioprosthesis. Because biological tissues used for implantablebioprostheses originate from non-autologous sources, the biologicaltissue must be treated prior to implantation to maintain a sufficientdegree of mechanical strength and dimensional stability. At the sametime, the biological tissue must also be treated to reduce itsantigenicity in the patient and to reduce actual and potential bindingsites for calcium.

Glutaraldehyde has long been the reagent of choice for cross-linking andsterilizing biological tissues for use in prosthetic heart valves. Theuse of glutaraldehyde, however, has many significant disadvantages.Because of its tendency to polymerize in solution, glutaraldehydefixation often results in the generation of aldehydes groups associatedwith the fixed tissue. Additionally, glutaraldehyde reacts with the freeamines in the tissue to generate labile Schiff bases. Both aldehydes andSchiff bases represent potential calcium binding sites that may lead tocalcification. Additionally, glutaraldehyde cross-linking affordslimited opportunities to tailor the properties of the cross-linkedtissue after fixation and provides little or no synthetic handles forthe rational design of a cross-linked network. Moreover, becauseglutaraldehyde is cytotoxic, it prevents the desired cellular in-growthand integration of the implanted and glutaraldehyde-treatedbioprosthetic tissue.

The present disclosure describes alternative methods for cross-linkingbiological tissue using an anchor compound and a difunctional linkingcompound, the anchor and difunctional linking compounds each comprisingcomplementary ones of a bio-orthogonal binding pair. The reactionbetween the bio-orthogonal binding pair has certain advantages overglutaraldehyde-based fixation. One advantage is that the reactionbetween the bio-orthogonal binding pair is highly specific to oneanother, thereby reducing or even eliminating the possibility ofundesired side reactions between any one of the bio-orthogonal bindingpair and tissue functional groups present in or native to biologicaltissue.

As used herein, “bio-orthogonal binding pair” refers to a pair offunctional groups which react with and couple one another. The reactionand coupling between complementary ones of the bio-orthogonal bindingpair is mutually exclusive such that each one of the bio-orthogonalbinding pair does not react with any tissue functional groups or withany functional groups found inside living systems.

As used herein, “tissue functional groups” refer to functional groupswhich are native to biological tissue and, more particularly, incollagenous tissue, such as, for example, cardiac valves, blood vessels,skin, dura mater, pericardium, small intestinal submucosa (“SIStissue”), ligaments and tendons. Exemplary tissue functional groupsinclude amines, hydroxyls, sulfhydryls, aldehydes, and carboxylic acids.

In a preferred embodiment, the bio-orthogonal binding pair comprises anazide and an acetylene. It is understood that the azide and acetylenegroups of the bio-orthogonal binding pair can be present as either aterminal or an internal group within an anchor compound or a linkingcompound used in accordance with the method. While the reaction of thebio-orthogonal binding pair itself is specific to one another, one orboth of the anchor compound and the linking compound can compriseadditional functional groups, such as those which react with tissuefunctional groups which can be reactive with other functional groups,such as tissue functional groups. However, it is understood that theadditional functional groups of the first or linking compound are notreactive with any one of the bio-orthogonal binding pair.

The reaction between the bio-orthogonal binding pair can take placeeither in the presence or absence of a catalyst. FIG. 1 depicts acopper-catalyzed reaction between an exemplary bio-orthogonal bindingpair comprising an azide and an alkyne functional group. As shown inFIG. 1, the reaction of an azide with an acetylene results in a cyclic1,4-disubstituted [1,2,3]-triazole. The inclusion of a copper catalystpermits this reaction to take place in an aqueous solution and at roomtemperature. In a preferred embodiment, the copper is a copper salt or acopper derivative.

In the context of cross-linking biological tissue, the biological tissueis contacted with an anchor compound comprising one of thebio-orthogonal binding pairs. In order to couple the anchor compoundonto the biological tissue, the anchor compound preferably comprises afirst functional group that forms a covalent bond with or otherwisecouples a tissue functional group associated with the biological tissue.In one embodiment, the one of the bio-orthogonal binding pairs and thefirst functional group can be located on terminal ends of the anchorcompound, which can be straight-chained or branched.

In a preferred embodiment, the biological tissue is not cross-linkedwith glutaraldehyde or any other aldehyde-containing agent. In aparticularly preferred embodiment, the biological tissue is cross-linkedusing only the anchor and difunctional linking compounds disclosedherein, in which the anchor comprises one of the bio-orthogonal bindingpair and the difunctional linking compound comprises the other one ofthe bio-orthogonal binding pair. In accordance with this embodiment, thefirst functional group of the anchor compound is not an aldehyde group.Preferably, the first functional group is also not a carboxylic acidgroup. In a further preferred embodiment, neither one of the anchor northe linking compound comprises an aldehyde or a carboxylic acid group.In one embodiment, the anchor compound is one or a combination of animidazole-1-sulfonyl azide and trifluoromethanesulfonyl azide.

Examples of first functional groups include isothiocyanate, isocyanate,sulfonyl chloride, aldehydes, carbodiimides, acyl azides, anhydrides,fluorobenzenes, carbonates, N-Hydroxysuccinimides (NHS), NHS esters,imidoesters, epoxides, fluorophenyl esters and are depicted in FIG. 4.The first functional groups can also include amines, carboxylic acidsand alcohols. The R represented in each of these structures can compriseone of the bio-orthogonal binding pair or a combination of a spacer andone of the bio-orthogonal binding pair.

FIGS. 5A and 5B depict the mechanism of cross-linking biological tissuesutilizing an anchor compound comprising one of the bio-orthogonalbinding pair and a linking compound comprising the other one of thebio-orthogonal binding pair. While FIGS. 5A and 5B depict thecross-linking being performed with the anchor compound comprising theazide and the linking compound comprising the di-functional alkyne, itis understood that the cross-linking can be performed with an anchorcompound comprising an alkyne and a linking compound comprising adi-functional azide. The linking compound is preferably homodifunctionalso as to prevent the linking compound from polymerizing.

As shown in FIGS. 5A and 5B, the azide is coupled to the tissue collagenfibers by way of a first functional group which couples with a tissuefunctional group to couple the anchor compound onto the biologicaltissue. The biological tissue that is coupled to the anchor compound isthen exposed to a linking compound comprising at least two functionalgroups, the two functional groups each comprising the other one of thebio-orthogonal binding pair. As depicted in FIGS. 5A and 5B, the linkingcompound is a di-functional alkyne, with the alkyne being located at theterminal ends of the linking compound.

Either one or both of the anchor and linking compounds can furthercomprise a spacer. In FIG. 5B, the di-functional alkyne is depicted ascomprising an alkyl spacer having a length n. The length of the spacercan be tailored based on the desired mechanical properties for theresulting cross-linked biological tissue. For example, longer spacerscan be provided to produce a more pliable or flexible cross-linkedtissue, whereas shorter spacers can be provided to produce a stiffercross-linked tissue. In a preferred embodiment, the spacer has a lengthof 10≥n≥3. The spacer can be selected from one or a combination of abranched or straight-chain saturated or unsaturated hydrocarbon or apolymer, such as a polyethylene glycol (PEG). The spacer can also be oneor a combination of a polymeric elastomer, such as a polyurethane, apolyisobutylene, and a polysiloxane, a polymeric carbohydrate, such as apolysaccharide, hyaluronic acid, dextran sulfate, and heparin.

The spacer can further comprise one or a combination of a bioactive or abiodegradable group. The biodegradable group can be one or a combinationof disulfides, polyesters, orthoesters, polyhydroxybutyric acid,poly(glycolide), poly(lactide) and copolymers of poly(glycolide) andpoly(lactide). The bioactive group can be incorporated to either promoteor repress specific cell interactions within the biological tissue orbetween the biological tissue and the host, once implanted. Thebiodegradable group, such as a disulfide, can be provided on the spacerto permit the ability to partially or completely dissolve or dissociatethe cross-linkages formed within the biological tissue at a desired timeafter implantation in the host. The ability to partially or completelydissolve or dissociate the cross-linkages can be desirable in order topermit cellular migration and in-growth such that the implantedbiological tissue becomes integrated with the host on a cellular level.The spacer can further include additional functional groups which can beused to couple or tether a specific drug or imaging molecule.

It is understood that the inclusion of additional functional groups onthe spacer, however, preferably does not comprise any functional groupsthat would be reactive with the biological tissue, the tissue functionalgroups or any one or both of the bio-orthogonal binding pair.

The reaction between the bio-orthogonal binding pair can be facilitatedwith a catalyst. Thus, the exposing of the biological tissue coupled tothe anchor compound to the linking compound can be performed in thepresence of a catalyst. Preferred catalysts include one or a combinationof a copper-based catalyst, a ruthenium-based catalyst and asilver-based catalyst. In another preferred embodiment, the catalystincludes one or a combination of a copper salt, a ruthenium salt and asilver salt. In a further preferred embodiment, the catalyst includesone or a combination of a copper-based derivative, a ruthenium-basedderivative and a silver-based derivative.

In a preferred embodiment, the catalyst is a Cu(I) catalyst. Because theCu(I) catalyst is cytotoxic, it has the advantage of also serving as asterilant for the cross-linked biological tissue. In embodiments where acytotoxic catalyst is used, the method further comprises rinsing thebioprosthetic tissue after the exposing step to eliminate or reduce thelevels of the cytotoxic catalyst to at or below aphysiologically-acceptable limit.

In one preferred embodiment, the rinsing can be performed using anaqueous, non-aqueous or anhydrous solutions. Aqueous solutions include asaline solution, preferably a buffered saline solution, such as aphosphate-buffered saline solution. Aqueous, non-aqueous or anhydroussolutions include glycerol solutions, polyethylene glycol (PEG)solutions, and ketone solutions, such as acetones. Treatment withcertain aqueous, non-aqueous or anhydrous solutions, such as thoseinvolving glycerol, permits the bioprosthetic tissue to be stored dry,i.e., in a manner that the tissue is not in contact with a liquidpreservative solution. In an alternative embodiment, the cross-linkingof the biological tissue can be performed in the absence of a catalyst.In this embodiment, the bio-orthogonal binding pair can comprise anazide and a cycloalkyne. The cycloalkyne is characterized as havingsufficient ring-strain to drive the cycloaddition reaction between theazide and the cycloalkyne at room temperature and without the need for acatalyst to drive the forward reaction.

In a preferred embodiment, the cycloalkyne has a ring strain of greaterthan 5 kcal/mol, more preferably greater than 10 kcal/mol and mostpreferably greater than 15 kcal/mol.

In another preferred embodiment, the cycloalkyne comprises one or moreelectron-withdrawing substituent. The one or more electron-withdrawingsubstituent preferably comprise one or more halogens, most preferablyfluorine. In a particularly preferred embodiment, the cycloalkyne is amono- or di-fluorinated cyclooctyne in which the electron-withdrawingfluorine substituents are located at the propargylic position.

FIG. 2 depicts the azide-di-fluorinated cyclooctyne cycloadditionreaction scheme in which the electron-withdrawing fluorine substituentsare located at the propargylic position. FIG. 6 further depicts alinking compound comprising two di-fluorinated cyclooctyne groups atterminal ends of an alkyl or polymer spacer having n length.

After treatment of the tissue with the anchor and linking compoundscomprising the bio-orthogonal binding pair, the tissue can be furthertreated to cap functional groups which play a role in tissuecalcification. Such functional groups can include aldehyde andcarboxylic acid groups on the native tissue or which result fromtreating or exposing the tissue with glutaraldehyde, formaldehyde orother aldehyde-containing compounds.

Thus, in one preferred embodiment, particularly in embodiments where thetissue is also treated with glutaraldehyde, formaldehyde and otheraldehyde-containing compounds, the process can further comprise atreatment with a capping and a reducing agent following the crosslinkingof the tissue using the anchor and difunctional linking compoundsdescribed above.

Insofar as the tissue may comprise residual aldehyde groups, the tissuecan be subjected to a capping process by contacting the tissue with acapping agent, such as ethanolamine, and a reducing agent, such assodium borohydride. Exemplary capping and reducing agents and processesare described in U.S. Pat. No. 7,972,376, the entire contents of whichare incorporated by reference, as if fully set forth herein.

Alternatively, tissue aldehydes can also be oxidized to carboxylic acidsand the carboxylic acids can be reacted with alcohols or amines

In yet further embodiments, tissue functional groups can be reacted withvarious nucleophiles and/or electrophiles in the presence of anappropriate catalyst, as described in U.S. patent application Ser. No.14/074,379, filed Nov. 7, 2013, the entire contents of which areincorporated herein by reference, as if fully set forth herein.

The bioprosthetic tissue can further undergo treatment with anhydrous,non-aqueous or aqueous glycerol solutions to substantially, if notcompletely, dehydrate the bioprosthetic tissue for dry storage.

In a preferred embodiment, the anhydrous or non-aqueous solutioncomprises glycerol and, more preferably, a solution of 75 wt % glyceroland 25 wt % ethanol, and the bioprosthetic tissue is soaked in theglycerol solution for at least one hour. The bioprosthetic tissue isthen removed and placed in a clean hood to allow removal of excesssolution.

In a preferred embodiment, the anhydrous or non-aqueous solution is asolution of glycerol and a C₁-C₃ alcohol, wherein the treatment solutioncomprises 60-95% by volume glycerol. Suitable treatment for thebiological tissues are described in U.S. Pat. No. 8,007,992, issued Aug.30, 2011, to Edwards Lifesciences Corp., the entire contents of whichare incorporated herein by reference as if fully set forth herein.

In another preferred embodiment, an aqueous glycerol solution can beused to at least partially dehydrate the tissue, as described in U.S.Pat. No. 6,534,004, issued Mar. 18, 2003, issued to The Cleveland ClinicFoundation, the entire contents of which are incorporated herein byreference in its entirety as if fully set forth herein.

The terms “dry” or “dehydrated,” as used herein, is understood toinclude residual treatment solution or moisture or humidity from theambient environment following treatment with the anhydrous, non-aqueousor aqueous glycerol solutions.

The dehydrated bioprosthetic tissue is provided in a sealed package,preferably in a double sterile barrier packaging consisting of a rigidtray (PETG) with a Tyvek lid. The sealed package preferably does notcontain a liquid preservative solution in contact with the tissue. Thepackage is sealed in a clean room, and sterilized in 100% ethyleneoxide.

While the present disclosure describes specific embodiments ofbio-orthogonal binding pairs, it is understood that it is not so limitedand that the disclosure encompasses any pair of functional groups whichengage in a mutually exclusive reaction and coupling with one another.Thus, it is to be understood that the detailed description and specificexamples, while indicating preferred embodiments of the presentinvention, are given by way of illustration and not limitation. Manychanges and modifications within the scope of the present invention canbe made without departing from the spirit thereof, and the disclosureincludes all such modifications.

What is claimed is:
 1. A cross-linked bioprosthetic tissue comprising: a biological tissue comprising one or more tissue functional groups; a plurality of anchor compounds, each of the plurality of the anchor compounds comprising a first functional group and a second functional group, the first functional group being coupled to one of the tissue functional groups of the biological tissue and the second functional group being one of a bio-orthogonal binding pair; and a plurality of linking compounds, each of the plurality of linking compounds comprising at least two functional groups, the at least two functional groups comprising the other one of the bio-orthogonal binding pair; wherein each one of the at least two functional groups of each linking compound is coupled to the second functional group of one of the plurality of anchor compounds to form cross-linkages within the biological tissue.
 2. The cross-linked bioprosthetic tissue of claim 1, wherein the bio-orthogonal binding pair comprises an azide and an acetylene.
 3. The cross-linked bioprosthetic tissue of claim 2, wherein the acetylene is incorporated in a cyclic compound having ring strain.
 4. The cross-linked bioprosthetic tissue of claim 3, wherein the acetylene is a cyclooctyne comprising one or more electron-withdrawing groups.
 5. The cross-linked bioprosthetic tissue of claim 4, wherein at least one of the one or more electron-withdrawing groups is a halogen.
 6. The cross-linked bioprosthetic tissue of claim 5, wherein the halogen is fluorine.
 7. The cross-linked bioprosthetic tissue of claim 2, wherein the first functional group of each of the plurality of anchor compounds is selected from the group consisting of: a carbodiimide, a fluorobenzene, and a fluorophenyl ester.
 8. The cross-linked bioprosthetic tissue of claim 1, wherein each tissue functional group is selected from the group consisting of: an amine, a hydroxyl, a sulfhydryl, a carbonyl, and a carboxylic acid.
 9. The cross-linked bioprosthetic tissue of claim 1, wherein either one or both of the anchor and the linking compounds comprises a spacer.
 10. The cross-linked bioprosthetic tissue of claim 9, wherein the spacer does not comprise functional groups that are reactive with the biological tissue, the tissue functional groups, or any one of the bio-orthogonal binding pair.
 11. The cross-linked bioprosthetic tissue of claim 9, wherein at least one of the plurality of linking compounds comprises the spacer, wherein the spacer comprises at least one of a polysaccharide and a dextran sulfate.
 12. The cross-linked bioprosthetic tissue of claim 9, wherein the spacer is selected from the group consisting of: branched or straight-chain saturated and unsaturated hydrocarbons.
 13. The cross-linked bioprosthetic tissue of claim 9, wherein the spacer comprises one or a combination of a bioactive and a biodegradable group.
 14. The cross-linked bioprosthetic tissue of claim 13, wherein: the spacer comprises the biodegradable group; and the biodegradable group is a disulfide.
 15. The cross-linked bioprosthetic tissue of claim 2, wherein the azide is present as an internal group within each of the plurality of anchor compounds and/or the acetylene is present as an internal group within the linking compound.
 16. The cross-linked bioprosthetic tissue of claim 1, wherein the first functional group of each of the plurality of anchor compounds is selected from the group consisting of: an isothiocyanate, an isocyanate, a sulfonyl chloride, an aldehyde, a carbodiimide, an acyl azide, an anhydride, a fluorobenzene, a carbonate, and a fluorophenyl ester.
 17. The cross-linked bioprosthetic tissue of claim 16, wherein each one of the at least two functional groups of each linking compound is coupled to the second functional group of one of the plurality of anchor compounds to form cross-linkages within the bioprosthetic tissue.
 18. The cross-linked bioprosthetic tissue of claim 17, wherein the bio-orthogonal binding pair comprises an azide and an acetylene.
 19. The cross-linked bioprosthetic tissue of claim 18, wherein: at least one of the plurality of anchor compounds comprises a spacer; wherein the spacer comprises a biodegradable group.
 20. The cross-linked bioprosthetic tissue of claim 18, wherein: either one or both of the plurality of anchor compounds and the plurality of linking compounds comprises a spacer; and wherein the spacer comprises at least one functional group coupled to an imaging molecule.
 21. The cross-linked bioprosthetic tissue of claim 18, wherein each of the plurality of anchor compounds and each of the plurality of linking compounds comprises a spacer. 